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Age-dependent decrease in the hepatic uptake of taurocholic acid resembles that for ouabain
Biochemical
Pharmacology.
ooo6-;1952/90 %3.00+0.00 @ 1990. Pergamon Press plc
Vol. 39, No. 7, pp. 1223-1228. 1990.
Printed in Great Britain.
AGE-DEPENDENT DECREASE IN THE HEPATIC UPTAKE OF TAUROCHOLIC ACID RESEMBLES THAT FOR OUABAIN A POSSIBLE
ROLE OF SURFACE
MEMBRANE
PROTEIN
MINORU OHTA and KENICHI KJTANI* First Laboratory of clinical Physiology, Tokyo Metropolitan Institute of gerontology,
NOBILITY
3.5-2, Sakaecho,
Itabashi-ku, Tokyo, Japan (Received
17 April 1989; accepted 9 November
1989)
Abstract-Isoiated hepatocytes were prepared from Wistar-derived male rats of different ages (4, 12 and 27-29 months) by the collagenase perfusion method. The hepatic uptake rate of taurocholate (TC) for the saturable fraction was calculated by subtracting the non-saturable fraction from the total hepatic uptake. The V,,, and the apparent affinity constant Km were computed for the saturable fraction by means of non-linear regression. The V,,, (nmolfmg protein/min, mean t SE) for young rats (N = 6) was 2.15 2 0.11, whereas in old rats (N = 4) the value was 50% lower (1.16 r 0.11, P < 0.005). in contrast, Km (,nM) values were not significantly different between young (25&? t 1.90) and old (30.34 _t 4.96) rats. There was a significant inverse linear relationship (r = 0.79; P < 0.01) between the age of rats and the uptake velocity (nmol/mg/mg protein/min) at 1 PM of TC, suggesting a steady and almost linear decrease of TC uptake velocity with age. The rate of decrease per month (2.1%) was quite close to the value for ouabain uptake (2.8%) previously found by the authors. Furthermore, a marked linearity was observed between the average values for TC uptake rates for three age groups and corresponding lateral diffusion constants of hepatocyte plasma membrane proteins previously obtained by the authors using the fluorescence recovery after photobleaching method, The results support our previous proposal that protein mobility within the hepatocyte surface membrane may play at least a partial role in regulation of carrier-mediated hepatocyte uptake functions for various materials.
The biliary excretion of iv. injected ouabain (a nonmetabol~zable neutral glycoside) decreases with age in an almost linear fashion in rats of both sexes [l31, largely owing to a steady decrease in the hepatic uptake of ouabain [3]. The lateral mobility of proteins (lateral diffusion constant) in the hepatocyte surface membrane, as measured by the fluorescence recovery after photobleaching (FRAP) technique, also decreases with age in a linear fashion, in both rats [2,4] and mice [S]. Furthermore, pretreatment with spironolactone (SP) was shown to increase both the biliary excretion of ouabain and the lateral diffusion constant of hepatocyte surface membrane proteins, in rats of three different ages [2]. Interestingly, SP pretreatment was reported to decrease (rather than increase) the lipid fluidity of hepatocyte surface membranes, as assessed by a fluorescence anisotropy method using diphenylhexatriene (DPH) [6,7]. On the basis of these studies, the working hypothesis has been proposed that protein mobility in the hepatocyte surface membrane as suggested from the results of our FRAP study, is a significant regulatory mechanism for carrier-mediated membrane functions, such as hepatic uptake of materials [2]. If our hypothesis is correct, the linear decrease with age in the hepatic uptake observed for ouabain may also exist for other materials having transport mechanisms different from that for ouabain. To test this hypothesis, in the present study we examined the hepatic uptake of taurocholic acid * To whom correspondence BP 39:~.F
should be addressed.
(TC) by isolated hepatocytes obtained from rats of different ages. TC was chosen because it is generally believed that the uptake mechanisms for the two substances are different. The receptors for TC and ouabain differ [g, 91, and TC uptake is mainly Na+ dependent [IO, 111, whereas ouabain uptake is Na+ independent [12,13]. MATERIALSANDMETHODS
Tritiated water (0.25 mCi/g), [carbonyl-‘4C]dextran (1.24 mCi/g, molecular weight 50,0~70,0~), and [carbonyl-i4C]taurocholic acid (46.7 mCi/ mmol) were purchased from New England Nuclear (Boston, MA). The possible contamination of the ]14C]dextran preparation by lower molecular weight materials which might have penetrated the hepatocytes was examined by means of ultrafiltration using Centricon membrane filters (Amicon, Danvers, MA) as previously reported [3]. It was judged that no correction for this factor was necessary. Sodium taurocholate (TC) was from the Sigma Chemical Co. (St Louis, MO). The chemical purity of this compound was more than 98%, as assessed by HPLC. As an incubation medium, the modified Waymouth MB752/1 medium (Gibco, Grand Island, NY) was added to 25 mM HEPES (Dojindo Laboratories, Kumamoto, Japan) as described previously [3]. The pH (7.4) and osmolality (300 mOsm/ kg) of the medium were adjusted to physiological levels by adding 1 N NaOH solution and NaCl, respectively. All chemical agents were of analytical grade.
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M. OWA
Male Wistar rats of three different ages (young, 4 months; middle-aged, 12 months; old, 27-29months) were used. These animals were bred and raised under specific pathogen-free (SPF) conditions at the aging farm of the institute. The husbandry condrtrons 1141, the pathological condition [15] observed in the latter period of their lives, and their survival rates 1151 have been reported elsewhere. Isolated hepatocytes were obtained from these rats by the collageaase perfusion method as described previously [3]. After washing, the hepatocytes were stored in suspension (about 2.0 X lO”ceIls/mL) at 4” in the incubation medium for about 2 hr before uptake study. TC uptake velocity determination was performed by the modified method as described by Iga and Klaassen 1161. The hepatocyte suspension (about 10 mt) was preincubated for 3 min at 37” under an atmosphere of 95% O2 and 5% CO2 with constant shaking (10 oscillations/min). One hundred microliters of incubation medium containing about 6-13 nCi of [ i4C]TC with unlabeled TC of nine different concentrations (l-750 FM) was placed in a OS-mL centrifuge tube containing 50 PL of 3 M NaOH solution in the bottom of a tube. covered by a 100 ,uL silicone layer with a density of 1.015 which was prepared from silicone oil (d = 1.05, Aldrich Chemical Company, Milwaukee, WI) and decahydronaphthalane (d = 0.876; Wako Pure Chemical Industry, Osaka, Japan) as reported previously 131. This silicone layer separated the TC fraction and NaOH layers before and during the incubation period. The centrifuge tube was preincubated for 3 min and 100 PI.. of hepatocyte suspension was added quickly to the TC solution. After incubation for 5, 10, 15, 20 or 25sec, hepatocytes were separated from the incubation medium and sedimented to the bottom of the tube by centrifugal filtration and lysed in the NaOH solution. The cell fraction Iysed in NaOH solution was added with Aquazol I1 (New England Nuclear). and the specific radioactivity was counted by a scintillation counter [3]. Uptake of TC by the hepatocytes was calculated from the specific radioactivity of cell fractions after appropriate correction for water adhering to the hepatocyte surface by using %I,0 and f “%Jdextran [3]. Using a cell preparation obtained from the liver of one animal, uptake rates for nine different concentrations were determined for each measurement. Hepatocytes viability was estimated by the Trypan blue exclusion test. Preparations with below 90% viability were not used for the experiments. The concentration of cellular protein was determined using a portion of the cell suspension used for each incubation experiment by the method of Lowry et al. [17]. One milliliter of hepatocyte suspension was washed three times with a 0.9% NaCI solution. After centrifugation, the cell pellets were lysed by addition of 8 mL of HZ0 and 2 ml, of a 5 M NaOH solution and were used for protein concentration determination. Bovine serum albumin was used as a standard. As is shown in Fig. 2, a non-linear relationship was obtained between the initial uptake velocity and bile salt concentrations. As the rate of uptake became linear with substrate concentrations above 100 FM for all studies, the overall uptake process was considered
and K. KITANI to be a combination of a saturable process and a linear (non-saturable) one mediated by a simple diffusion, as reported in previous studies [9-11,131. The rate of hepatic uptake of TC from the saturable fraction was obtained by dissociating a saturable fraction from a non-saturable fraction. To separate the relative contribution of each of these processes, an apparent diffusion constant(k) for the non-saturable fraction, was calculated from the slopes of the linear portion of each curve above 100 @I by applying the least-squares linear regression analysis. The hepatic uptake rate of the non-saturable fraction at each concentration of TC (i.e., k times concentration) was subtracted from the overall uptake rate at each concentration to yield the initial velocity of the saturable component. V,,,,, and K,,, were calculated from the relations between the initial uptake rate of the saturable fraction and each TC concentration in the medium by means of a nonlinear regression as described by Wjlkinson 1181with minor modifications for a Vax-11 computer. We then selected the best fit to the Michaelis-Menten equation. statistical analysis for kinetic parameters was performed by the method of Cleland 1191. The ieastsquares linear regression analysis was also applied to evaluate the relation between the uptake rate at a given concentration and rat age. For all studies, P values lower than 0.05 were judged to be significant. RESULTS
Figure 1 shows examples of the sequential changes in the hepatic uptake of TC at several different concentrations as examined in hepatocyte preparations from a young rat. The uptake was linear against time up to 25 set over a wide range of TC concentrations in both young and old rats. From the slope of the linear portion of the hepatic uptake, the rate of initial hepatic uptake was calculated. Figure 2 shows an example of the relationship between the uptake rate of TC and its concentration in the medium. The relationship can be analysed as a sum of saturable and non-saturable components for TC uptake. After subtraction of the non-saturable fraction from the total uptake, the relation between the uptake rate and the concentration in the medium for the saturable fraction followed the MichaelisMenten kinetics. Figure 3 shows the saturation kinetics for TC uptake by isolated hepatocyte prepared from young and aid rats, using TC uptake rates of the saturable component. The V,,,,, values (nmol/mg protein/min, mean rt SE) calculated from these relations were 2.15 to.11 (N = 6), 1.45 + 0.32 (N -4). and 1.16 +- 0.11 (N = 4) for young, middle-aged and old rats, respectively. The V,,,, value in old rats was significantly lower than that in yaung rats (P < 0.005). In contrast. K,,, (pM) values were not signi~cantly different among the different age groups (P > 0.5) (25.88 c 1.90, 30.34 rt 1.45, 30.34 t 4.96 for young, middle aged and old rats, respectively). Figure 4 shows the relationship between the initial uptake velocity of TC by hepatocytes (u) and the age of animals (X) at a TC concentration of 1 @M. A signi~cant linear relationship (Y = 77. I1 - I .597Z r = -0.788; P < 0.005) was obtained. The rate of
Hepatic uptake of TC
1225
1.
/
/ i-
, /
/ /
10 Time,
20
30
set
Fig. 1. Time course of TC uptake at various taurocholate concentrations by isolated hepatocytes prepared from a young Wistar rat.
10 t
0
100
250 Taurocholate concentration,
500
750
j&l
Fig. 2. An example of the relation between the initial TC uptake rates and TC concentrations by isolated hepatocytes prepared from a young male Wistar rat. (A) Total initial uptake rates measured directly from the cell suspension radioactivity. (A) Calculated uptake rates for the saturable fraction. Values were obtained by subtracting the uptake rate of the non-saturable fraction from the total uptake rate. Uptake rates of the non-saturable fraction were obtained as the products of the diffusion constant (k), obtained from the linear regression of the original values above 100,~~Min TC concentration and corresponding TC concentrations shown as broken line.
decrease in uptake velocity of 2.1% per month was obtained by the calculation. Figure 5A shows the relationship between TC uptake velocity at 1 PM (Y) and the lateral diffusion constant of hepatocyte surface membrane proteins (X) as previously determined by the FRAP technique [Z], in three corresponding age groups. A marked linearity between the two independently (Y’ was found determined parameters -107.66 + 67.52X, r = 0.984). Figure 5B shows the same relationship for ouabain uptake velocity previously found by the authors [2,3]
r = 0.978). (Y = -115.15 + 63.70X, marked linearity can be observed.
Again,
a
DISCUSSION
The results of the present study have shown that the initial uptake rate of TC for the saturable fraction by isolated rat hepatocytes decreases in an almost linear fashion with age, as previously shown for ouabain uptake [3]. The rate of decrease per month for the hepatic uptake rate of TC (2.1%) was surprisingly close to the value for ouabain (2.8%) previously found in our laboratory [3]. Furthermore,
OHTA and K. K~I‘ANI
Vmax 2.15
I
Km
25.88
+
0.11 +
1.90
Vmax 1.16 + 0.1 t 30.34 ?: 4.96
0
50 Taurocholate
l+-zz100 concentration, @I
Fig. 3. Kinetic studies of initial hepatic uptake rates of TC in young (4 months, 0, N = 6) and old (27-29 months, 0, N = 4) rat groups for the saturable fraction of TC uptake. Vertical bar indicates SD. Kinetic parameters were calculated by a non-linear regression analysis. Values are presented as mean i SE. N indicates the number of rats in each age group.
20.
r=
-0.78836
.
P < 0.005 0
IO Age
.
20
30
months
Fig. 4. The relationship between the initial uptake velocity of TC by hepatocytes (Y) and the age of animals (X) at a TC concentration in the medium of 1 PM.
the 50% decrease in V,,, with unaltered Km value in old rats compared with values in young rats found in the present experiment for TC also agrees with our previous study on ouabain uptake kinetics [3]. Generally speaking, the V,,,,, of a carrier-mediated transport system is considered to be the function of the number of carrier unit and the dif~sion constant of the carrier-ligand complex [20]. The decrease in the V,, value with unaltered Km is therefore interpreted as owing to a decrease in either the carrier unit number or the diffusion constant (or to a combination of both). If the transport system requires
an expenditure of energy, the decrease in energy may also affect the if,,,;,,. If the decline in the hepatic uptake rate of TC is related to alteration of Na+,K’-ATPase activity during aging. the decline in the uptake rate for ouabain must therefore be explained in terms of a mechanism totally different from that of TC. because ouabain uptake is Na’ independent 112, 131. If this is the case, the agreement of the degree of r/,,,, decrease with age for the two substances may be only a coincidence. Alternatively, the V,,,i,, decrease in TC uptake may be due to an age-dependent decrease in the carrier unit number or the mobility of the carrier-ligand complex 1201. However, the carrier systems postulated for these materials in previous reports remain a matter of controversy. Until recently, it had been relatively well established that the carrier for bile salts (anions) uptake is different From that for ouabain, a neutral comp~~und ]X, 9.211. However, a recent study by Petzinger et al. (221 has suggested that these two different ~omp~)unds may share a common transport protein for their hepatic uptake processes. If indeed, the carrier proteins for TC and ouabain are the same, as maintained by Petzinger et al. [22], the comparable decrease in V,,:,, values for these two substances during aging might be most easily explained on the basis of the decrease in their common carrier unit numbers. At present. however, it is not clear whether the carrier proteins for TC and ouabain are really the same. It appears rather unlikely that these two materials completely share the same carrier, because the devel~~p;l~entai changes in the hepatic uptakes of these materials are totally different 123,241. If they are different. therefore, the V,,, decrease should be explained as the result of the independent decrease in numbers of the respective (and different) carrier units for the two materials. Again, the agreement of the magnitude of their decreases should be a coincidental phenomenon. A third possibility. that the decrease in the diffusion constant of the carrier-hgand complex during aging is the cause, requires special consideration. because we have previously shown that the lateral mobility of hepatocyte surface membrane proteins as assessed by FRAP technique, decreases in a linear fashion with rat age [2,4]. Although membrane proteins previously studied by our FRAP technique are a complex of proteins bound to oxidized ribo~avin [2.5.26], and are not specific proteins directly mediating the transports of TC and ouabain, there ISsome theoretical and experimental basis that the mobilities of cell surface membrane proteins are generally more restricted as the age advances [4.27-291. If. indeed. the membrane protein movement is progressively restricted during the aging process by the decrease in passive permeability of the membrane for potassium [4,27,28], cross-linking of membrane proteins ]28] and or changes in lipid composition. it is not unreasonable to assume that the mobility of the membrane proteins involved with the transports of materials are aiso affected during aging as suggested by our previous FRAP studies [2,4,5]. Assuming that the mobilities of both carrier proteins are similarly altered during aging, as suggested by our previous studies 141,and that such an alteration regulates
1227
Hepatic uptake of TC (81
(A)
fouabain)
(taurocholate)
Y = -115.15 + 83.70x
Y = -107.66 * 67.52x 2s
*f 75
_z Q 3” 0 % ti 50 *CD f E 0. o”a g E, 2s
0
1.8 Diffusion constant
10-%m~ lsec
2.0
Diffusion constant
2.5 to-%rn* lsec
Fig. 5. The relationship
between TC uptake velocity at 1 yM and the diffusion constant of hepatocyte surface membrane proteins, as previously de&ermined by our FRAP method [Zj in three different age groups (A) and a similar relationship for ouabain uptake rate (B). The latter figure comprises the results of previous studies of ouabain uptake [3] and FRAP study [Z].
the hepatic uptake of materials, our results on both TC and ouabain uptake in aging rat livers can be more plausibly explained as owing to a common mechanism. Surprisingly linear and similar relations found between the uptakes for these materials and the latera diffusion constant of hepatocyte surface membrane proteins, as assessed by our FRAP system (Fig. SA, B), are consistent with our working hypothesis that the changes in membrane protein mobility that occur during aging may be a significant factor for regulating the hepatic uptake rates for many different materials that are mediated by membranelocated protein carriers. Acknowledge~enrs-The efficient secretarial work of Mrs K. Tagami is gratefully acknowledged. REFERENCES 1. Kitani K, Kanai S. Miura R, Morita Y and Kasahara
M, The effect of aging on the biliary excretion of ouabain in the rat. Exe GerontoE 13: 9-17. 1978. 2. Kitani K, Zs-Nagy I, ‘Kanai S, Sato Y and Ohta M, Correlation between the bihary excretion of ouabain and the lateral mobility of hepatocyte plasma membrane proteins in rhe rat. The‘effects of age and spironolactone pretreatment. Henatolo,qy ._. 8: 125-131, 1988. . 3. Ohta M, Kanai S. Sato Y and Kitani K. Aae-dependent decrease in the hepatic uptake and biliary excretion of ouabain in rats. B~uc~e~ Pharma~o~37: 935-942.1988. 4. Zs-Nagy I, Kitani K, Ohta M, 2%Nagy V and Imahori K, Age-dependent decrease of the lateral diffusion constant of proteins in the plasma membrane of hepatocytes as revealed by fluorescence recovery after photobleaching in tissue smears. Arch Gerontol Geriatr 5: 131-146. 1986. 5. Zs-Nagy I, Kitani K and Ohta M, Age-dependence
of the lateral mobility of proteins in the plasma membrane of hepatocytes of C57BL/6 mice: FRAP studies on liver smears. J Gerontoi Biol Sci 44: B83-87, 1989. 6. Miner Jr PB, Sneller M and Crawford SS, Spironalactone and ~nrenone-induced changes in hepatic (Na+,K‘)ATPase activity, surface membrane cholesterol and phospholipid, and fluorescence polarization in the rat. Hepatofogy 3: 481-488, 1983. 7. Smith DJ and Gordon ER, Role of liver plasma membrane ~uidity in the pathogen~is of cholestasis (Abstract). ~epaco~ogy 6: 771, 1986.
8. Schanker LS, Secretion of organic compounds in bile. In: Handbook of Physiof (Ed. Code CF), pp. 24332449. America1 Physiological Society, Washington DC, 1968. 9. Anwer MS and Hegner D, Effect of Na+ on bile acid uptake by isolated rat hepatocytes. Evidence for a heterogenous system. Hoppe-Seylers Z Physiol Chem 359: 181-192, 1978. 10. Schwarz LR, Burr B, Schwenk M, Pfaff E and Greim
H, Uptake of taurocholic acid into isolated rat livercells. Fur J B~ochem 55: 617-623. 1975. 11. Dietmaier A, Gasser R, Graf J and Peterhk M, Investigations on the sodium dependence of bile acid fluxes in the isolated perfused liver. Biochim Biophys Acra 4413: 81-91, 12.
1976.
Eaton DL and Klaassen CD, Carrier-mediated port of ouabain in isolated hepatocytes.
transI Pharma~ol
Exp Ther 205: 480-488, 1978. 13. Meijer DFK, Vonk RJ, Scholtens EJ and Levine WG,
The influence of dehydrocbolate on hepatic uptake and biliarv excretion of 3H-taurocholate and 3H-ouabain. Drug-Metab Dispos 4: 1-7, 1976. 14. Nokubo M. Phvsical-chemical and biochemical
differences in hver plasma membranes in aging F-344 rats. J Gerontol40: 409-414, 1985. 15. Inoue T, Kanisawa M, Kuramoto K and Oota K, Life spans and morphologicai findings of Wistar and F-344 rats bred with standard or restricted feeding. Biomed Gerontol6: 4-5, 1982. 16. Iga T and Klaassen CD, Uptake of bile acids by isolated rat hepatocytes. Biochem Pharmacol 31: 211-216, 1982. 17. Lowry OH, Rosebrough NJ, Farr AJ and Randall RJ,
Protein measurement with the Fohn ohenol reaaent. J Biol Chem 193: 265-275, 1951. ’ y 18. WiIkinson GN. Statistical estimations in enzvme kinetics. Bioc&m J SO: 324-332, 1961. 19. Cleland WW, The statistical analysis of enzyme kinetic data. Ado Enzymol29: l-32, 1967. 20. de Pont JJHHM and Bonting SL, Permeability of membranes. In: Mammalian Cell Membranes, Val4, Mem” branes and Cellular ~u~ctio~ (Eds. Jamieson CA and Robinson DM), pp. 116-144. Butte~orths, London, 1977. .. 21. Eaton DL and Richards JA, Kinetic evaluation of carrier-mediated transport of ouabain and taurochohc acid in isolated rat hepatocytes. Evidence for independent transport systems. Biochem Pharmacoi 35: 27X-2725, 1986. 22. Petzinger E, Fischer K and Fasold H, Role of the bile
1228
M. OHTA and K. KITANI
acid transport system in hepatoceliularouabain uptake. In : Cardiac ~~y~osides jr785-i 985. 3~o~h~~~y-Phar~acol~gy-~~~niea~ Rebvance (Eds. Erdmann E, Creef K and Skou JC), pp. 297-304. Steinkopff Darmstadt, Springer, New York, 1986. 23. Klaassen CD, Independence of bile acid and ouabain uptake: studies in the new born rat. Proc Snc Exp Biol Med 157: 64-69, 1978. 24. Stacey NH and Kfaassen CD, Uptake of ouabain by isolated hepatocytes from livers of developing rats. J Pharmucol Exp Ther 211: 360-363, 1979. 25. Nokubo M, Zs-Nagy I+ Kitani K and Ohta M, Characterization of peroxide-induced autofluorescence of rat liver plasma membranes. Biochim Biophys Acta 939: 441-448, 1988.
26. Nokubo M, Ohta M, Kitani K and Zs-Nagy I, Identifkation of protein-bound ribo~avi~ in rat hepatocyte plasma membrane as a source of autofluorescence. ~~0~~~~ ~iop~~s Acra 981: 30>308, 1989. 27. Zs-Nagy I, A membrane hypothesis of aging. J Theor I%0175: 189195. 1978. 28. Zs-Nagy I, An attempt to answer the questions of theoretical gerontology on the basis of the membrane hypothesisof aging. Ado Biosciences64: 393-413,1987. 29. Zs-Nagy I and Nagy K, On the role of cross-linking of celiular proteins in aging, Me& Age&g Dee I4: 245 251. 1980.
Pharmacology.
ooo6-;1952/90 %3.00+0.00 @ 1990. Pergamon Press plc
Vol. 39, No. 7, pp. 1223-1228. 1990.
Printed in Great Britain.
AGE-DEPENDENT DECREASE IN THE HEPATIC UPTAKE OF TAUROCHOLIC ACID RESEMBLES THAT FOR OUABAIN A POSSIBLE
ROLE OF SURFACE
MEMBRANE
PROTEIN
MINORU OHTA and KENICHI KJTANI* First Laboratory of clinical Physiology, Tokyo Metropolitan Institute of gerontology,
NOBILITY
3.5-2, Sakaecho,
Itabashi-ku, Tokyo, Japan (Received
17 April 1989; accepted 9 November
1989)
Abstract-Isoiated hepatocytes were prepared from Wistar-derived male rats of different ages (4, 12 and 27-29 months) by the collagenase perfusion method. The hepatic uptake rate of taurocholate (TC) for the saturable fraction was calculated by subtracting the non-saturable fraction from the total hepatic uptake. The V,,, and the apparent affinity constant Km were computed for the saturable fraction by means of non-linear regression. The V,,, (nmolfmg protein/min, mean t SE) for young rats (N = 6) was 2.15 2 0.11, whereas in old rats (N = 4) the value was 50% lower (1.16 r 0.11, P < 0.005). in contrast, Km (,nM) values were not significantly different between young (25&? t 1.90) and old (30.34 _t 4.96) rats. There was a significant inverse linear relationship (r = 0.79; P < 0.01) between the age of rats and the uptake velocity (nmol/mg/mg protein/min) at 1 PM of TC, suggesting a steady and almost linear decrease of TC uptake velocity with age. The rate of decrease per month (2.1%) was quite close to the value for ouabain uptake (2.8%) previously found by the authors. Furthermore, a marked linearity was observed between the average values for TC uptake rates for three age groups and corresponding lateral diffusion constants of hepatocyte plasma membrane proteins previously obtained by the authors using the fluorescence recovery after photobleaching method, The results support our previous proposal that protein mobility within the hepatocyte surface membrane may play at least a partial role in regulation of carrier-mediated hepatocyte uptake functions for various materials.
The biliary excretion of iv. injected ouabain (a nonmetabol~zable neutral glycoside) decreases with age in an almost linear fashion in rats of both sexes [l31, largely owing to a steady decrease in the hepatic uptake of ouabain [3]. The lateral mobility of proteins (lateral diffusion constant) in the hepatocyte surface membrane, as measured by the fluorescence recovery after photobleaching (FRAP) technique, also decreases with age in a linear fashion, in both rats [2,4] and mice [S]. Furthermore, pretreatment with spironolactone (SP) was shown to increase both the biliary excretion of ouabain and the lateral diffusion constant of hepatocyte surface membrane proteins, in rats of three different ages [2]. Interestingly, SP pretreatment was reported to decrease (rather than increase) the lipid fluidity of hepatocyte surface membranes, as assessed by a fluorescence anisotropy method using diphenylhexatriene (DPH) [6,7]. On the basis of these studies, the working hypothesis has been proposed that protein mobility in the hepatocyte surface membrane as suggested from the results of our FRAP study, is a significant regulatory mechanism for carrier-mediated membrane functions, such as hepatic uptake of materials [2]. If our hypothesis is correct, the linear decrease with age in the hepatic uptake observed for ouabain may also exist for other materials having transport mechanisms different from that for ouabain. To test this hypothesis, in the present study we examined the hepatic uptake of taurocholic acid * To whom correspondence BP 39:~.F
should be addressed.
(TC) by isolated hepatocytes obtained from rats of different ages. TC was chosen because it is generally believed that the uptake mechanisms for the two substances are different. The receptors for TC and ouabain differ [g, 91, and TC uptake is mainly Na+ dependent [IO, 111, whereas ouabain uptake is Na+ independent [12,13]. MATERIALSANDMETHODS
Tritiated water (0.25 mCi/g), [carbonyl-‘4C]dextran (1.24 mCi/g, molecular weight 50,0~70,0~), and [carbonyl-i4C]taurocholic acid (46.7 mCi/ mmol) were purchased from New England Nuclear (Boston, MA). The possible contamination of the ]14C]dextran preparation by lower molecular weight materials which might have penetrated the hepatocytes was examined by means of ultrafiltration using Centricon membrane filters (Amicon, Danvers, MA) as previously reported [3]. It was judged that no correction for this factor was necessary. Sodium taurocholate (TC) was from the Sigma Chemical Co. (St Louis, MO). The chemical purity of this compound was more than 98%, as assessed by HPLC. As an incubation medium, the modified Waymouth MB752/1 medium (Gibco, Grand Island, NY) was added to 25 mM HEPES (Dojindo Laboratories, Kumamoto, Japan) as described previously [3]. The pH (7.4) and osmolality (300 mOsm/ kg) of the medium were adjusted to physiological levels by adding 1 N NaOH solution and NaCl, respectively. All chemical agents were of analytical grade.
1223
1224
M. OWA
Male Wistar rats of three different ages (young, 4 months; middle-aged, 12 months; old, 27-29months) were used. These animals were bred and raised under specific pathogen-free (SPF) conditions at the aging farm of the institute. The husbandry condrtrons 1141, the pathological condition [15] observed in the latter period of their lives, and their survival rates 1151 have been reported elsewhere. Isolated hepatocytes were obtained from these rats by the collageaase perfusion method as described previously [3]. After washing, the hepatocytes were stored in suspension (about 2.0 X lO”ceIls/mL) at 4” in the incubation medium for about 2 hr before uptake study. TC uptake velocity determination was performed by the modified method as described by Iga and Klaassen 1161. The hepatocyte suspension (about 10 mt) was preincubated for 3 min at 37” under an atmosphere of 95% O2 and 5% CO2 with constant shaking (10 oscillations/min). One hundred microliters of incubation medium containing about 6-13 nCi of [ i4C]TC with unlabeled TC of nine different concentrations (l-750 FM) was placed in a OS-mL centrifuge tube containing 50 PL of 3 M NaOH solution in the bottom of a tube. covered by a 100 ,uL silicone layer with a density of 1.015 which was prepared from silicone oil (d = 1.05, Aldrich Chemical Company, Milwaukee, WI) and decahydronaphthalane (d = 0.876; Wako Pure Chemical Industry, Osaka, Japan) as reported previously 131. This silicone layer separated the TC fraction and NaOH layers before and during the incubation period. The centrifuge tube was preincubated for 3 min and 100 PI.. of hepatocyte suspension was added quickly to the TC solution. After incubation for 5, 10, 15, 20 or 25sec, hepatocytes were separated from the incubation medium and sedimented to the bottom of the tube by centrifugal filtration and lysed in the NaOH solution. The cell fraction Iysed in NaOH solution was added with Aquazol I1 (New England Nuclear). and the specific radioactivity was counted by a scintillation counter [3]. Uptake of TC by the hepatocytes was calculated from the specific radioactivity of cell fractions after appropriate correction for water adhering to the hepatocyte surface by using %I,0 and f “%Jdextran [3]. Using a cell preparation obtained from the liver of one animal, uptake rates for nine different concentrations were determined for each measurement. Hepatocytes viability was estimated by the Trypan blue exclusion test. Preparations with below 90% viability were not used for the experiments. The concentration of cellular protein was determined using a portion of the cell suspension used for each incubation experiment by the method of Lowry et al. [17]. One milliliter of hepatocyte suspension was washed three times with a 0.9% NaCI solution. After centrifugation, the cell pellets were lysed by addition of 8 mL of HZ0 and 2 ml, of a 5 M NaOH solution and were used for protein concentration determination. Bovine serum albumin was used as a standard. As is shown in Fig. 2, a non-linear relationship was obtained between the initial uptake velocity and bile salt concentrations. As the rate of uptake became linear with substrate concentrations above 100 FM for all studies, the overall uptake process was considered
and K. KITANI to be a combination of a saturable process and a linear (non-saturable) one mediated by a simple diffusion, as reported in previous studies [9-11,131. The rate of hepatic uptake of TC from the saturable fraction was obtained by dissociating a saturable fraction from a non-saturable fraction. To separate the relative contribution of each of these processes, an apparent diffusion constant(k) for the non-saturable fraction, was calculated from the slopes of the linear portion of each curve above 100 @I by applying the least-squares linear regression analysis. The hepatic uptake rate of the non-saturable fraction at each concentration of TC (i.e., k times concentration) was subtracted from the overall uptake rate at each concentration to yield the initial velocity of the saturable component. V,,,,, and K,,, were calculated from the relations between the initial uptake rate of the saturable fraction and each TC concentration in the medium by means of a nonlinear regression as described by Wjlkinson 1181with minor modifications for a Vax-11 computer. We then selected the best fit to the Michaelis-Menten equation. statistical analysis for kinetic parameters was performed by the method of Cleland 1191. The ieastsquares linear regression analysis was also applied to evaluate the relation between the uptake rate at a given concentration and rat age. For all studies, P values lower than 0.05 were judged to be significant. RESULTS
Figure 1 shows examples of the sequential changes in the hepatic uptake of TC at several different concentrations as examined in hepatocyte preparations from a young rat. The uptake was linear against time up to 25 set over a wide range of TC concentrations in both young and old rats. From the slope of the linear portion of the hepatic uptake, the rate of initial hepatic uptake was calculated. Figure 2 shows an example of the relationship between the uptake rate of TC and its concentration in the medium. The relationship can be analysed as a sum of saturable and non-saturable components for TC uptake. After subtraction of the non-saturable fraction from the total uptake, the relation between the uptake rate and the concentration in the medium for the saturable fraction followed the MichaelisMenten kinetics. Figure 3 shows the saturation kinetics for TC uptake by isolated hepatocyte prepared from young and aid rats, using TC uptake rates of the saturable component. The V,,,,, values (nmol/mg protein/min, mean rt SE) calculated from these relations were 2.15 to.11 (N = 6), 1.45 + 0.32 (N -4). and 1.16 +- 0.11 (N = 4) for young, middle-aged and old rats, respectively. The V,,,, value in old rats was significantly lower than that in yaung rats (P < 0.005). In contrast. K,,, (pM) values were not signi~cantly different among the different age groups (P > 0.5) (25.88 c 1.90, 30.34 rt 1.45, 30.34 t 4.96 for young, middle aged and old rats, respectively). Figure 4 shows the relationship between the initial uptake velocity of TC by hepatocytes (u) and the age of animals (X) at a TC concentration of 1 @M. A signi~cant linear relationship (Y = 77. I1 - I .597Z r = -0.788; P < 0.005) was obtained. The rate of
Hepatic uptake of TC
1225
1.
/
/ i-
, /
/ /
10 Time,
20
30
set
Fig. 1. Time course of TC uptake at various taurocholate concentrations by isolated hepatocytes prepared from a young Wistar rat.
10 t
0
100
250 Taurocholate concentration,
500
750
j&l
Fig. 2. An example of the relation between the initial TC uptake rates and TC concentrations by isolated hepatocytes prepared from a young male Wistar rat. (A) Total initial uptake rates measured directly from the cell suspension radioactivity. (A) Calculated uptake rates for the saturable fraction. Values were obtained by subtracting the uptake rate of the non-saturable fraction from the total uptake rate. Uptake rates of the non-saturable fraction were obtained as the products of the diffusion constant (k), obtained from the linear regression of the original values above 100,~~Min TC concentration and corresponding TC concentrations shown as broken line.
decrease in uptake velocity of 2.1% per month was obtained by the calculation. Figure 5A shows the relationship between TC uptake velocity at 1 PM (Y) and the lateral diffusion constant of hepatocyte surface membrane proteins (X) as previously determined by the FRAP technique [Z], in three corresponding age groups. A marked linearity between the two independently (Y’ was found determined parameters -107.66 + 67.52X, r = 0.984). Figure 5B shows the same relationship for ouabain uptake velocity previously found by the authors [2,3]
r = 0.978). (Y = -115.15 + 63.70X, marked linearity can be observed.
Again,
a
DISCUSSION
The results of the present study have shown that the initial uptake rate of TC for the saturable fraction by isolated rat hepatocytes decreases in an almost linear fashion with age, as previously shown for ouabain uptake [3]. The rate of decrease per month for the hepatic uptake rate of TC (2.1%) was surprisingly close to the value for ouabain (2.8%) previously found in our laboratory [3]. Furthermore,
OHTA and K. K~I‘ANI
Vmax 2.15
I
Km
25.88
+
0.11 +
1.90
Vmax 1.16 + 0.1 t 30.34 ?: 4.96
0
50 Taurocholate
l+-zz100 concentration, @I
Fig. 3. Kinetic studies of initial hepatic uptake rates of TC in young (4 months, 0, N = 6) and old (27-29 months, 0, N = 4) rat groups for the saturable fraction of TC uptake. Vertical bar indicates SD. Kinetic parameters were calculated by a non-linear regression analysis. Values are presented as mean i SE. N indicates the number of rats in each age group.
20.
r=
-0.78836
.
P < 0.005 0
IO Age
.
20
30
months
Fig. 4. The relationship between the initial uptake velocity of TC by hepatocytes (Y) and the age of animals (X) at a TC concentration in the medium of 1 PM.
the 50% decrease in V,,, with unaltered Km value in old rats compared with values in young rats found in the present experiment for TC also agrees with our previous study on ouabain uptake kinetics [3]. Generally speaking, the V,,,,, of a carrier-mediated transport system is considered to be the function of the number of carrier unit and the dif~sion constant of the carrier-ligand complex [20]. The decrease in the V,, value with unaltered Km is therefore interpreted as owing to a decrease in either the carrier unit number or the diffusion constant (or to a combination of both). If the transport system requires
an expenditure of energy, the decrease in energy may also affect the if,,,;,,. If the decline in the hepatic uptake rate of TC is related to alteration of Na+,K’-ATPase activity during aging. the decline in the uptake rate for ouabain must therefore be explained in terms of a mechanism totally different from that of TC. because ouabain uptake is Na’ independent 112, 131. If this is the case, the agreement of the degree of r/,,,, decrease with age for the two substances may be only a coincidence. Alternatively, the V,,,i,, decrease in TC uptake may be due to an age-dependent decrease in the carrier unit number or the mobility of the carrier-ligand complex 1201. However, the carrier systems postulated for these materials in previous reports remain a matter of controversy. Until recently, it had been relatively well established that the carrier for bile salts (anions) uptake is different From that for ouabain, a neutral comp~~und ]X, 9.211. However, a recent study by Petzinger et al. (221 has suggested that these two different ~omp~)unds may share a common transport protein for their hepatic uptake processes. If indeed, the carrier proteins for TC and ouabain are the same, as maintained by Petzinger et al. [22], the comparable decrease in V,,:,, values for these two substances during aging might be most easily explained on the basis of the decrease in their common carrier unit numbers. At present. however, it is not clear whether the carrier proteins for TC and ouabain are really the same. It appears rather unlikely that these two materials completely share the same carrier, because the devel~~p;l~entai changes in the hepatic uptakes of these materials are totally different 123,241. If they are different. therefore, the V,,, decrease should be explained as the result of the independent decrease in numbers of the respective (and different) carrier units for the two materials. Again, the agreement of the magnitude of their decreases should be a coincidental phenomenon. A third possibility. that the decrease in the diffusion constant of the carrier-hgand complex during aging is the cause, requires special consideration. because we have previously shown that the lateral mobility of hepatocyte surface membrane proteins as assessed by FRAP technique, decreases in a linear fashion with rat age [2,4]. Although membrane proteins previously studied by our FRAP technique are a complex of proteins bound to oxidized ribo~avin [2.5.26], and are not specific proteins directly mediating the transports of TC and ouabain, there ISsome theoretical and experimental basis that the mobilities of cell surface membrane proteins are generally more restricted as the age advances [4.27-291. If. indeed. the membrane protein movement is progressively restricted during the aging process by the decrease in passive permeability of the membrane for potassium [4,27,28], cross-linking of membrane proteins ]28] and or changes in lipid composition. it is not unreasonable to assume that the mobility of the membrane proteins involved with the transports of materials are aiso affected during aging as suggested by our previous FRAP studies [2,4,5]. Assuming that the mobilities of both carrier proteins are similarly altered during aging, as suggested by our previous studies 141,and that such an alteration regulates
1227
Hepatic uptake of TC (81
(A)
fouabain)
(taurocholate)
Y = -115.15 + 83.70x
Y = -107.66 * 67.52x 2s
*f 75
_z Q 3” 0 % ti 50 *CD f E 0. o”a g E, 2s
0
1.8 Diffusion constant
10-%m~ lsec
2.0
Diffusion constant
2.5 to-%rn* lsec
Fig. 5. The relationship
between TC uptake velocity at 1 yM and the diffusion constant of hepatocyte surface membrane proteins, as previously de&ermined by our FRAP method [Zj in three different age groups (A) and a similar relationship for ouabain uptake rate (B). The latter figure comprises the results of previous studies of ouabain uptake [3] and FRAP study [Z].
the hepatic uptake of materials, our results on both TC and ouabain uptake in aging rat livers can be more plausibly explained as owing to a common mechanism. Surprisingly linear and similar relations found between the uptakes for these materials and the latera diffusion constant of hepatocyte surface membrane proteins, as assessed by our FRAP system (Fig. SA, B), are consistent with our working hypothesis that the changes in membrane protein mobility that occur during aging may be a significant factor for regulating the hepatic uptake rates for many different materials that are mediated by membranelocated protein carriers. Acknowledge~enrs-The efficient secretarial work of Mrs K. Tagami is gratefully acknowledged. REFERENCES 1. Kitani K, Kanai S. Miura R, Morita Y and Kasahara
M, The effect of aging on the biliary excretion of ouabain in the rat. Exe GerontoE 13: 9-17. 1978. 2. Kitani K, Zs-Nagy I, ‘Kanai S, Sato Y and Ohta M, Correlation between the bihary excretion of ouabain and the lateral mobility of hepatocyte plasma membrane proteins in rhe rat. The‘effects of age and spironolactone pretreatment. Henatolo,qy ._. 8: 125-131, 1988. . 3. Ohta M, Kanai S. Sato Y and Kitani K. Aae-dependent decrease in the hepatic uptake and biliary excretion of ouabain in rats. B~uc~e~ Pharma~o~37: 935-942.1988. 4. Zs-Nagy I, Kitani K, Ohta M, 2%Nagy V and Imahori K, Age-dependent decrease of the lateral diffusion constant of proteins in the plasma membrane of hepatocytes as revealed by fluorescence recovery after photobleaching in tissue smears. Arch Gerontol Geriatr 5: 131-146. 1986. 5. Zs-Nagy I, Kitani K and Ohta M, Age-dependence
of the lateral mobility of proteins in the plasma membrane of hepatocytes of C57BL/6 mice: FRAP studies on liver smears. J Gerontoi Biol Sci 44: B83-87, 1989. 6. Miner Jr PB, Sneller M and Crawford SS, Spironalactone and ~nrenone-induced changes in hepatic (Na+,K‘)ATPase activity, surface membrane cholesterol and phospholipid, and fluorescence polarization in the rat. Hepatofogy 3: 481-488, 1983. 7. Smith DJ and Gordon ER, Role of liver plasma membrane ~uidity in the pathogen~is of cholestasis (Abstract). ~epaco~ogy 6: 771, 1986.
8. Schanker LS, Secretion of organic compounds in bile. In: Handbook of Physiof (Ed. Code CF), pp. 24332449. America1 Physiological Society, Washington DC, 1968. 9. Anwer MS and Hegner D, Effect of Na+ on bile acid uptake by isolated rat hepatocytes. Evidence for a heterogenous system. Hoppe-Seylers Z Physiol Chem 359: 181-192, 1978. 10. Schwarz LR, Burr B, Schwenk M, Pfaff E and Greim
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Eaton DL and Klaassen CD, Carrier-mediated port of ouabain in isolated hepatocytes.
transI Pharma~ol
Exp Ther 205: 480-488, 1978. 13. Meijer DFK, Vonk RJ, Scholtens EJ and Levine WG,
The influence of dehydrocbolate on hepatic uptake and biliarv excretion of 3H-taurocholate and 3H-ouabain. Drug-Metab Dispos 4: 1-7, 1976. 14. Nokubo M. Phvsical-chemical and biochemical
differences in hver plasma membranes in aging F-344 rats. J Gerontol40: 409-414, 1985. 15. Inoue T, Kanisawa M, Kuramoto K and Oota K, Life spans and morphologicai findings of Wistar and F-344 rats bred with standard or restricted feeding. Biomed Gerontol6: 4-5, 1982. 16. Iga T and Klaassen CD, Uptake of bile acids by isolated rat hepatocytes. Biochem Pharmacol 31: 211-216, 1982. 17. Lowry OH, Rosebrough NJ, Farr AJ and Randall RJ,
Protein measurement with the Fohn ohenol reaaent. J Biol Chem 193: 265-275, 1951. ’ y 18. WiIkinson GN. Statistical estimations in enzvme kinetics. Bioc&m J SO: 324-332, 1961. 19. Cleland WW, The statistical analysis of enzyme kinetic data. Ado Enzymol29: l-32, 1967. 20. de Pont JJHHM and Bonting SL, Permeability of membranes. In: Mammalian Cell Membranes, Val4, Mem” branes and Cellular ~u~ctio~ (Eds. Jamieson CA and Robinson DM), pp. 116-144. Butte~orths, London, 1977. .. 21. Eaton DL and Richards JA, Kinetic evaluation of carrier-mediated transport of ouabain and taurochohc acid in isolated rat hepatocytes. Evidence for independent transport systems. Biochem Pharmacoi 35: 27X-2725, 1986. 22. Petzinger E, Fischer K and Fasold H, Role of the bile
1228
M. OHTA and K. KITANI
acid transport system in hepatoceliularouabain uptake. In : Cardiac ~~y~osides jr785-i 985. 3~o~h~~~y-Phar~acol~gy-~~~niea~ Rebvance (Eds. Erdmann E, Creef K and Skou JC), pp. 297-304. Steinkopff Darmstadt, Springer, New York, 1986. 23. Klaassen CD, Independence of bile acid and ouabain uptake: studies in the new born rat. Proc Snc Exp Biol Med 157: 64-69, 1978. 24. Stacey NH and Kfaassen CD, Uptake of ouabain by isolated hepatocytes from livers of developing rats. J Pharmucol Exp Ther 211: 360-363, 1979. 25. Nokubo M, Zs-Nagy I+ Kitani K and Ohta M, Characterization of peroxide-induced autofluorescence of rat liver plasma membranes. Biochim Biophys Acta 939: 441-448, 1988.
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